Streptococcus oligofermentans Inhibits Streptococcus mutans in Biofilms at Both Neutral pH and Cariogenic Conditions Xudong Bao1,2, Johannes Jacob de Soet2, Huichun Tong3, Xuejun Gao1, Libang He4, Cor van Loveren2, Dong Mei Deng2* 1 Department of Cariology and Endodontology, Peking University School and Hospital of Stomatology, Beijing, China, 2 Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands, 3 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, 4 State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China * [email protected]



OPEN ACCESS Citation: Bao X, de Soet JJ, Tong H, Gao X, He L, van Loveren C, et al. (2015) Streptococcus oligofermentans Inhibits Streptococcus mutans in Biofilms at Both Neutral pH and Cariogenic Conditions. PLoS ONE 10(6): e0130962. doi:10.1371/journal.pone.0130962 Editor: Zezhang Wen, LSU Health Sciences Center School of Dentistry, UNITED STATES Received: September 20, 2014 Accepted: May 27, 2015 Published: June 26, 2015 Copyright: © 2015 Bao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Homeostasis of oral microbiota can be maintained through microbial interactions. Previous studies showed that Streptococcus oligofermentans, a non-mutans streptococci frequently isolated from caries-free subjects, inhibited the cariogenic Streptococcus mutans by the production of hydrogen peroxide (HP). Since pH is a critical factor in caries formation, we aimed to study the influence of pH on the competition between S. oligofermentans and S. mutans in biofilms. To this end, S. mutans and S. oligofermentans were inoculated alone or mixed at 1:1 ratio in buffered biofilm medium in a 96-well active attachment model. The single- and dual-species biofilms were grown under either constantly neutral pH or pHcycling conditions. The latter includes two cycles of 8 h neutral pH and 16 h pH 5.5, used to mimic cariogenic condition. The 48 h biofilms were analysed for the viable cell counts, lactate and HP production. The last two measurements were carried out after incubating the 48 h biofilms in buffers supplemented with 1% glucose (pH 7.0) for 4 h. The results showed that S. oligofermentans inhibited the growth of S. mutans in dual-species biofilms under both tested pH conditions. The lactic acid production of dual-species biofilms was significantly lower than that of single-species S. mutans biofilms. Moreover, dual-species and single-species S. oligofermentans biofilms grown under pH-cycling conditions (with a 16 h low pH period) produced a significantly higher amount of HP than those grown under constantly neutral pH. In conclusion, S. oligofermentans inhibited S. mutans in biofilms not only under neutral pH, but also under pH-cycling conditions, likely through HP production. S. oligofermentans may be a compelling probiotic candidate against caries.

Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors received no specific funding for this work. Competing Interests: The authors have declared that no competing interests exist.

PLOS ONE | DOI:10.1371/journal.pone.0130962 June 26, 2015

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Introduction The human oral cavity harbours a dynamic microbial community, which consists of more than 700 bacterial species [1]. In the healthy situation, this community maintains a healthy microbial homeostasis, through a dynamic balance of synergistic and antagonistic microbial interactions. Disturbance of this homeostasis can lead to shifts in microbial composition and eventually cause diseases e.g. dental caries [2]. Dental caries is chemical dissolution of the dental hard tissues by the acid produced when bacteria metabolise dietary carbohydrates. Under prolonged low-pH environment, the outgrowth of acidogenic and aciduric species such as mutans streptococci and lactobacilli shifts the microbial composition and enhances the dissolution of the dental hard tissue, eventually causing caries [3,4]. One of the key concepts in the prevention and treatment of dental caries is to prevent disturbance of or to re-establish a healthy microbial homeostasis [3]. Clinical studies have indicated that in the dental biofilms high numbers of non-mutans streptococci, such as Streptococcus sanguinis and Streptococcus gordornii, were often associated with low numbers of cariogenic bacteria Streptococcus mutans and this association was mostly observed in healthy subjects, while the inverse was typically found in subjects with caries [5,6]. These findings have emphasised the potential of caries prevention via modulating oral microbial ecology. Among the commensal oral non-mutans streptococci, Streptococcus oligofermentans has several interesting traits. It was frequently isolated from caries-free subjects or healthy non-carious tooth surfaces [7,8]. It produced less acid from glucose than S. mutans [8] and inhibited the growth of S. mutans [9]. With molecular techniques, Tong et al [9] demonstrated that S. oligofermentans inhibited the growth of S. mutans through the production of hydrogen peroxide (HP) both in suspensions and in biofilms. S. oligofermentans employs three types of enzymes, pyruvate oxidase (POX), lactate oxidase (LOX) and L-amino acid oxidase, to produce HP [9,10,11]. The synergistic action of POX and LOX maximized the HP production of S. oligofermentans [10]. The ability of S. oligofermentans producing HP from lactic acid is particularly interesting, since lactic acid is the major organic acid produced by dental biofilms. This trait of S. oligofermentans may provide dual benefits: minimising pH drop by converting lactic acid into HP and inhibiting the cariogenic bacteria S. mutans through HP production. Therefore, S. oligofermentans may be a good probiotic candidate for maintaining healthy oral microflora. Although several studies have reported that S. oligofermentans could inhibit the growth of S. mutans in a dual-species biofilm [8,10,11], some characteristics of the biofilm model used in these studies may limit the clinical relevance of their findings: firstly, the studied biofilms were “bottom-biofilms”. These biofilms mostly contain sedimented cells, which are not incorporated by active attachment, while active attachment is a prerequisite for oral biofilm formation. Secondly, the pH in the bottom-biofilm model was unknown and not controlled. Environmental factors, such as the presence of oxygen, sugar availability and pH, were shown to greatly affect the HP production of S. oligofermentans, thereby its inhibition on the growth of S. mutans [12,13]. A previous study demonstrated that the inhibitory effect of S. oligofermentans on S. mutans decreased with the decreasing pH value. At pH 5.5, no interaction between two species was observed in the agar competition assay [12]. Since S. mutans was known to be aciduric and to be able to outcompete other bacterial species at cariogenic condition (pH 5.5), the above findings seemed to suggest the limitations of S. oligofermentans in maintaining healthy microflora at cariogenic conditions. As only planktonic cultures were tested in the previous study, it is relevant to re-evaluate the influence of pH in a biofilm model that allows bacterial active attachment. The aims of this study are to establish a pH-controllable active-attachment biofilm model and to explore the competition between S. mutans and S. oligofermentans in biofilms under two different pH conditions, constantly neutral pH and pH-cycling. The pH-cycling included a

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period of 8 h at neutral pH and a period of 16 h at pH 5.5, with the intention to mimic cariogenic conditions that dental biofilms often encounter.

Materials and Methods Bacterial strains and growth conditions The strains used in this study were Streptococcus mutans UA159 and Streptococcus oligofermentans LMG22279 [7]. Both bacterial strains were grown anaerobically (90% N2, 5% CO2, 5% H2) at 37°C. Biofilms were grown in a modified semi-defined biofilm medium (BM), which contains 10 mM (NH4)2SO4, 35 mM NaCl, 2 mM MgSO47H2O and was supplemented with filtersterilised vitamins (0.04 mM nicotinic acid, 0.1 mM pyridoxine HCl, 0.01 mM pantothenic acid, 1 μM riboflavin, 0.3 μM thiamine HCl, and 0.05 μM D-biotin), amino acids (4 mM L-glutamic acid, 1 mM L-arginine HCl, 1.3 mM L-cysteine HCl, and 0.1 mM L-tryptophan), and 0.3% (wt/vol) yeast extract [14]. To prepare BM of pH 7.0, 76 mM K2HPO4 and 15 mM KH2PO4 were added to the medium. To prepare BM of pH 5.5, 30 mM MES buffer was added to the medium. For pre-cultures, BMG was prepared by adding 0.4% of glucose to BM and for biofilm growth, BMS was prepared by adding 0.2% of sucrose to BM. This sucrose concentration was chosen because it could promote biofilm formation without causing pH changes.

Biofilm growth Biofilms were grown in an active attachment model [15]. This model consists of a standard 96-well microtiter plate and a lid with an identical number of polystyrene pegs that fit into wells (Nunc, Roskilde, Denmark). This model was chosen to examine exclusively the actively adhered biofilms, instead of bacterial sedimentation in the 96-well microtiter plate, and to retain the 96-well high-throughput advantage for testing multiple variables. Single or dualspecies biofilms were grown for 48 h before further analysis. In detail, to grow single-species biofilms, overnight (16 h) S. mutans and S. oligofermentans cultures in BMG were diluted to a final OD600nm of 0.04 in fresh BMS (pH 7.0) and 200 μl of each cell suspension was dispensed into a 96-well plate. To grow dual-species biofilms, the overnight cultures of each strain were diluted to a final OD600nm of 0.08 in BMS (pH 7.0) and mixed at 1:1 ratio before dispensing 200 μl into the 96-well plate. This mixture contains 4.6 x 106 (± 2.7 x 106) CFU/ml of S. mutans and 1.7 x 107 (± 7.4 x106) CFU/ml of S. oligofermentans. The plate was then covered with the lid containing pegs and incubated for 8 h. Thereafter, the pegs were rinsed with sterile distilled water to remove non-adherent bacterial cells. After rinsing, half of the pegs were inserted in BMS of pH 7.0, while the other half was inserted in BMS of pH 5.5, and both were further incubated for 16 h. Subsequently all pegs were rinsed with sterile distilled water and inserted again in BMS of pH 7.0. After another 8 h incubation, the pegs were inserted in either BMS of pH 7.0 or BMS of pH 5.5 for another 16 h. The 48 h biofilms formed on the pegs were collected for viable cell counts and were examined for their capability to produce lactic acid and HP. The schema of biofilm growth is illustrated in Fig 1. In each experiment, a total of 6 groups (single-/dual-species groups, grown under neutral pH or pH-cycling condition) were tested. Each group generally contained 8 biofilms replicates. Four replicates were used for viable cell counts and four were used for the measurement of lactic acid and HP production. The experiment was repeated 3 times.

Viable cell counts Each individual peg with biofilms was carefully cut off with a sterile scalpel without disturbing the biofilms and placed in 1ml CPW (5 g yeast extract, 1 g peptone, 8.5 g NaCl, 0.5 g L-cysteine

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Fig 1. The schema of the biofilm experiment. doi:10.1371/journal.pone.0130962.g001

hydrochloride per liter, adjusted to pH 7.3). Biofilms were dispersed by sonication on ice 60 times for 1 s at an amplitude of 40 W (Vibra cell, Sonics & Materials Inc., USA). Undiluted samples or serially diluted samples (100 μl) were plated onto Brain Heart Infusion (BHI) agar plates. The plates were incubated for 3 d. The colonies were counted and recorded as Colony Forming Unit (CFU). Since the morphologies of S. mutans and S. oligofermentans were distinct on BHI agar plates, the CFUs of S. mutans and S. oligofermentans in the dual-species biofilm samples were recorded separately based on the colony morphology. The images of the colonies are provided in S1 and S2 Figs. The detection limit of this viable cell count method is 100 CFU per sample.

HP and lactic acid quantification The capability of HP and lactic acid production of 48 h biofilms was examined by inserting the pegs with biofilms into a 96-well plate, filled with 200 μl per well buffered assay medium (pH 7.0) at 37°C for 4 h. The assay medium contained most of the components of BM except yeast extract, since no further biofilm growth was desired during the incubation. Glucose (1%) was added to the assay medium in order to trigger lactic acid production. After incubation, 50 μl assay medium was immediately used for HP measurement. The rest of the medium was stored at -20°C for lactic acid quantification. HP was quantified by an enzymatic assay with modifications [16]. In detail, 50 μl of culture medium was added to 45 μl of solution containing 2.5 mM 4-aminoantipyrine (4-amino-2,

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3-dimethyl-1-phenyl-3-pyrazolin-5-one; Sigma-Aldrich, St. Louis, MO, USA) and 0.17 M phenol in a 96-well plate. This reaction mixture was incubated for 5 min at room temperature; thereafter horseradish peroxidase (Sigma-Aldrich, St. Louis, MO, USA) was added at a concentration of 640 mU ml-1 in 0.2 M potassium phosphate buffer (pH 7.2). After 4 min, the absorbance was recorded at 510 nm in a spectrophotometer (Perkin Elmer, Norwalk, CT, USA). HP concentration of each sample was calculated from a standard curve generated with known concentrations of HP (Sigma-Aldrich, St. Louis, MO, USA). Lactic acid was measured with an enzymatic-spectrophotometric method [17]. The principle of the method is based on the enzymatic conversion of L-lactate to pyruvate with concomitant conversion of NAD to NADH, the increase in absorbance at 340 nm being proportional to NADH formation.

HP production of S. oligofermentans suspension cells at pH 7.0 or pH 5.5 To understand how environmental pH influenced the ability of HP production of S. oligofermentans, we chose to carry out the experiment on suspension cells since these cells were more homogenous and better controlled than the biofilm cells. Overnight S. oligofermentans culture grown in BMG (pH 7.0) was centrifuged and resuspended in either BM pH7.0 or BM pH5.5, without any addition of sucrose. The cell density was adjusted to an OD600nm of 0.9. The resuspensions were incubated anaerobically for either 4 or 16 h. The ability of these suspension cells to produce HP was examined in the same way as the biofilms were tested. In detail, the resuspensions were centrifuged, incubated in the buffered assay medium for 4 h. The supernatants were used for HP quantification (the procedure is described above). The S. oligofermentans cells before and after 4 or 16 h of incubation were also subjected to viable cell counts. This experiment was repeated 3 times. Duplicate samples per group were included in each experiment.

Statistical analysis The data were analysed with the Statistical Package for Social Science (SPSS, Version 17.0, Chicago, IL, USA). One-way ANOVA was used to evaluate the differences in CFU, HP and lactic acid concentration between single- and dual-species biofilms. Specifically, independent-samples t test was used to examine the influence of pH (neutral pH vs. pH-cycling) on the variables. The CFU counts were log transformed before the statistical tests. p

Streptococcus oligofermentans Inhibits Streptococcus mutans in Biofilms at Both Neutral pH and Cariogenic Conditions.

Homeostasis of oral microbiota can be maintained through microbial interactions. Previous studies showed that Streptococcus oligofermentans, a non-mut...
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